The determination of particular nucleic acid molecules, or expression of these molecules, is an extremely important facet of analytical and clinical chemistry. A vast number of different nucleic acid assays are known in the field. All of these assays may be said to have a common goal, i.e., the identification of particular nucleic acid molecules in samples. Achievement of this aim permits one to identify infections, such as bacterial or viral infections, to type tissues, to identify individuals (so-called "DNA fingerprinting"), and so forth.
One of the problems in nucleic acid assays is that the target materials, i.e., a particular nucleic acid molecule, exists as only one or very few copies. Thus, there has been a great deal of interest in purifying nucleic acid molecules so that the chances of actually finding the desired molecule is maximized.
Classic techniques have been developed for purifying nucleic acid molecules. One of the most basic of these is the method described by Maniatis et al., in Molecular Cloning: A Laboratory Manual (New York, Cold Spring Harbor Laboratory, 1982, pp. 280-1). This method teaches the lysis of target cells, using proteases, followed by phenol/chloroform extraction. The method takes a very long time to complete, and involves the use of hazardous, carcinogenic substances. Some of the problems associated with this method are discussed in Miller et al., WO89/07603 (Aug. 24, 1989), incorporated by reference. Further, the phenol/chloroform based methodologies all require the use of an alcohol precipitating agent, such as ethanol or isopropanol to separate nucleic acid molecules from their solvent. Risk of damage to the desired material, as well as loss of it, is very great.
The need for continued improvement in this very basic technology can be seen via the large number of patents and non-patent publications directed to it. These references are directed to improvements in obtaining desired nucleic acid molecules. They evidence the many different approaches one may take.
U.S. Pat. No. 5,231,015 to Cummins, e.g., teaches the use of a metal ion in lysis solutions. The ion is a cofactor for naturally occurring nucleic acid molecule polymerases. The theory is that the metal ions improves the inherent ability of native polymerases to copy the nucleic acid molecule of interest. Such a technique is especially useful in amplification methodologies, elaborated infra. U.S. Pat. No. 5,130,423, to van Ness et al., alleges improvements in the use of phenyl derivatives, such as benzyl alcohol, in the extraction of DNA. U.S. Pat. No. 5,128,247 to Koller is along the same lines, and teaches the use of chaotropic agents to lyse cells, followed by treatment with sulfated polysaccharide proteins, such as heparin.
U.S. Pat. No. 5,010,183 to Macfarlane, advises the art to use cationic detergents to purify nucleic acids, while U.S. Pat. No. 4,908,318, teaches detergent based lysis followed by solubilizing the nucleic acid molecules, and then spooling these.
U.S. Pat. No. 5,284,940 to Lin, et al. suggests the use of transferrin, globin, or serum albumin to prevent the action of polymerase inhibitors during an amplification reaction.
Most of the patents discussed supra deal with the preparation of samples for subsequent use in amplification assays, especially the very well known polymerase chain reaction, or "PCR" technique. This methodology, described in Mullis et al., U.S. Pat. No. 4,683,202, and incorporated by reference herein, shows how one can obtain multiple copies of a desired nucleic acid molecule via the use of one or two nucleic acid molecule primers, together with a polymerase, such as Thermus aquaticus, or "Taq" polymerase. The procedure by which the amplified nucleic acid molecules are obtained, however, is essentially that of Maniatis et al. The desire to improve this methodology can be seen, e.g., in Casareale et al., "Improved Blood Sample Processing For PCR" in PCR Methods and Applications (Cold Spring Harbor Laboratories, New York, 1992, pp. 149-153), incorporated by reference. The paper discusses the inherent limitations on PCR, including small sample volume, inhibition of application, sample stability, and so forth. Casareale et al. report improvements in the isolation of nucleic acids by using heat and detergents. The detergent used was Nonidet P-40, the chemical name of which is ethyl phenol poly(ethylene glycolether).sub.n, where "n" is usually a whole number of about 11. The success is attributed to a reduction in inhibition of DNA amplification. Others have made similar assertions, for example, in Ehrlich et al., ed. PCR Technology: Principles & Applications for DNA Amplification pp. 19-21, the use of Nonidet P-40, in combination with Tween 20 (Poly(oxyethylene).sub.n -sorbitane-monolaurate, when "n" is usually 20) is said to prevent inhibition of Taq polymerase in lysed samples where sodium dodecyl sulfate (SDS) was used in the lysing agent. The effect of the SDS is to inhibit any polymerases used in the amplification process. The detergent combination is alleged to alleviate the problem, when DNA and Mg.sup.2+ (a cofactor for Taq polymerase) are present, at 37.degree. C.
The use of detergents to neutralize SDS is, in a theoretical sense, not surprising. Haselbeck et al, "Studies on the effect of the Incubation Conditions, Various Detergents and Protein Concentration on the Enzymatic Activity of N-Glycosidase F (Glycopeptidase F), and Endoglycosidase F", in Topics In Biochemistry 8: 1-4 (1988), discuss the general inhibitory effect of SDS on enzymes. Haselbeck et al., then show that either NP-40 or Triton X-100 (octylphenolpoly(ethylene glycol ether).sub.n where "n" is about 10), inhibit the effect of SDS on the listed enzymes. Generalizations are not made and, indeed, as will be discussed, infra, broad generalizations in fact cannot be made as to the effect of detergents on eliminating the impact of SDS on amplification processes.
The body of art discussed supra points to one of the problems addressed by the invention. In brief, the agents used to lyse cells, and thereby free nucleic acids for amplification, frequently include sodium dodecyl sulfate, or "SDS". SDS however, has the undesired side effect of inhibiting the polymerases necessary to carry out amplification reactions.
Another problem in the art is the need to obtain very pure nucleic acid samples in as brief an amount of time as is possible. When cells are lysed, materials other than the target nucleic acid molecules are released, and these must be deemed contaminants. In the case of whole blood samples, there is a particularly serious problem caused by the voluminous amount of protein released, relative to the amount of nucleic acids. Included in these proteins are polymerase inhibitors. Porphyrin ring containing compounds, especially heme and its derivatives, are notoriously well known as polymerase inhibitors. Clearly, it is very important to remove these materials from samples.
The alcohol precipitation approach, discussed supra, is one way of removing nucleic acid molecules from impurities. Applicants will not repeat the drawbacks of this approach again. It would be desirable if one could quickly, and efficiently obtain pure samples of nucleic acid molecules without the need for an alcohol precipitation step. It is even more desirable to have such a method available where the nucleic acid molecules thus purified could be used immediately in an amplification process.
In U.S. Pat. No. 5,294,681 to Krupey, which is incorporated by reference in its entirety, a family of water insoluble, cross-linked polyhydroxy polycarboxylic acid molecules are described. These molecules are: ##STR1## wherein one carbonyl group of at least one maleoyl moiety thereof in each strand is covalently linked to a EQU -HN[H).sub.p (CH.sub.2)(OH).sub.m ]NH-moiety
to provide the presence therein of at least one cross linking moiety of the formula: ##STR2## wherein R is hydrogen or lower alkylene or lower alkoxy of 1-4 carbon atoms, or phenyl, z is an integer of 1-4, p is 0 or an integer up to z-1, m is 1 or an integer up to z, wherein the ratio of cross-links to poly (alkylene carbonic acid) strands is between 1 and about 200 to 2 are described as being useful for recovering proteins from aqueous media. The molecules sequester any proteins in the sample. A product based upon this patent, known as PROCIPITATE, is commercially available. The commercial product does not, however, adumbrate the particular acids used therein, but only refers to the patent.
Krupey describes the use of his novel molecules in the separation of DNA from proteins generally; however, the methods are only described generally, and always refer to the use of aqueous guanidium thiocyanate, a chaotrope, and the lysing agent. These methodologies are all reported in the context of methods where nucleic acid precipitation via the use of, e.g., alcohols, is also used.
Thus, there is another problem in the art in that the desirability of separating proteins is linked to the precipitation of DNA, which is not desirable.
It has now been found, first of all, that generalizations regarding detergent based inactivation of SDS cannot be made in the context of preparing samples for nucleic acid amplification. This is especially true for non-ionic detergents, where it has been found that phenyl group containing detergents, such as the "Triton" family of detergents, do not function to inhibit sodium dodecyl sulfate. Thus, one aspect of the invention is based upon the surprising recognition that non-ionic detergents which do not contain a phenyl group can be used alone, to inhibit sodium dodecyl sulfate, thus permitting improved purification of nucleic acid samples from whole cells.
A second aspect of the invention, also surprising, is that cross linked, polyhydroxy polycarboxylic acids of formula ##STR3## wherein one carbonyl group of at least one maleoyl moiety thereof in each strand is covalently linked to a EQU --HN[H).sub.p (CH.sub.2)(OH).sub.m ]NH-moiety
to provide the presence therein of at least one cross linking moiety of the formula: ##STR4## wherein R is hydrogen or lower alkylene or lower alkoxy of 1-4 carbon atoms, or phenyl, z is an integer of 1-4, p is 0 or an integer up to z-1, m is 1 or an integer up to z, wherein the ratio of cross-links to poly (alkylene carbonic acid) strands is between 1 and about 200 to 2 can be used in methods for purifying nucleic acids, where an alcohol precipitation step may be left out. Use of these compounds also inhibit SDS, and thus may be used alone or together with the detergents discussed supra.
These methods may be employed separately, or together.
These, and other aspect of the invention, will be seen in the disclosure which follows.